Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.     

A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.)

Two large-scale ethylmethanesulfonate (EMS) mutant populations from barley (Hordeum vulgare L.) cv. Optic have been developed to promote both forward and reverse genetics in this crop. Leaf material and seed from approximately 20 000 M(2) plants were individually harvested, freeze-dried and archived. DNA was isolated from 9216 plants from the 20 and 30 mm EMS treatments and assembled into 1152 eight-plant pools. To facilitate PCR-based mutation scanning an approach has been employed that combines cleavage of heteroduplexes using the Cel nuclease (Cel I), post-cleavage intercalating dye labeling and the subsequent detection of cleaved products on a Transgenomic WAVE-HS. The populations were evaluated by screening for induced mutations in two genes of interest and the induced mutations were validated by sequence analysis. To enhance the screening process, 12-16 M(3) progeny from each of the M(2) plants were assessed for visible phenotypes and the data entered into a web accessible database (http://bioinf.scri.sari.ac.uk/distilling/distilling.html).     

Growth factors regulate the survival and fate of cells derived from human neurospheres

Cells isolated from the embryonic, neonatal, and adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of both multipotent stem cells and more restricted progenitor populations. Neurospheres can also be generated from the embryonic human brain and in some cases have been expanded for extended periods of time in culture. However, the mechanisms controlling the number of neurons generated from human neurospheres are poorly understood. Here we show that maintaining cell-cell contact during the differentiation stage, in combination with growth factor administration, can increase the number of neurons generated under serum-free conditions from 8% to > 60%. Neurotrophic factors 3 and 4 (NT3, NT4) and platelet-derived growth factor (PDGF) were the most potent, and acted by increasing neuronal survival rather than inducing neuronal phenotype. Following differentiation, the neurons could survive dissociation and either replating or transplantation into the adult rat brain. This experimental system provides a practically limitless supply of enriched, non-genetically transformed neurons. These should be useful for both neuroactive drug screening in vitro and possibly cell therapy for neurodegenerative diseases.     

Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.     

Cloning, chromosomal sublocalization of the human soluble aminopeptidase P gene (XPNPEP1) to 10q25.3 and conservation of the putative proton shuttle and metal ligand binding sites with XPNPEP2

Human soluble ("cytosolic") aminopeptidase P (hsAmP) is an aminoacylprolyl hydrolase (EC 3.4.11.9) present in all tissues yet examined. hsAmP is related in terms of catalytic specificity to an ectoenzyme, membrane aminopeptidase P (hmAmP), which is largely limited in distribution to endothelia and brush border epithelia. Although both enzymes can degrade oligopeptides having N-terminal Xaa-Pro- moieties, hsAmP and hmAmP are of relatively low sequence homology. Recently, it has been shown that the two enzymes are not products of splice variants of the same gene. How hsAmP relates to hmAmP has clinical significance in that both can inactivate bradykinin, and AmP deficiency states have been described. The hmAmP gene (XPNPEP2) is disposed at chromosome Xq25, a disposition with clear meaning in terms of inheritance of hmAmP deficiencies. To further explore similarities and differences between hsAmP and hmAmP, the present study was begun to determine the chromosomal disposition of the hsAmP gene. Here we show that the gene is sublocalized on chromosome 10q25.3. We also show that hsAmP and hmAmP contain homologous blocks of sequence common to members of the "pita bread-fold" protein family, of which Escherichia coli methionine aminopeptidase is the prototype. The prototype is known to contain a proton shuttle and five divalent metal ligands, counterparts of which we identify in the homologous blocks of sequence in both hsAmP and hmAmP and compare to E. coli aminopeptidase.     

GOLAC: an endogenous anion channel of the Golgi complex

The Golgi complex is present in every eukaryotic cell and functions in posttranslational modifications and sorting of proteins and lipids to post-Golgi destinations. Both functions require an acidic lumenal pH and transport of substrates into and by-products out of the Golgi lumen. Endogenous ion channels are expected to be important for these features, but none has been described. Ion channels from an enriched Golgi fraction cleared of transiting proteins were incorporated into planar lipid bilayers. Eighty percent of the single-channel recordings revealed the same anion channel. This channel has novel properties and has been named GOLAC (Golgi anion channel). The channel has six subconductance states with a maximum conductance of 130 pS, is open over 95% of the time, and is not voltage-gated. Significant for Golgi function, the channel conductance is increased by reduction of pH on the lumenal surface. This channel may serve two nonexclusive functions: providing counterions for the acidification of the Golgi lumen by the H(+)-ATPase and removal of inorganic phosphate generated by glycosylation and sulfation of proteins and lipids in the Golgi.     

Secondary reduction mammaplasty: is using a different pedicle safe?

Reduction mammaplasty is a frequently performed procedure and one with consistent patient satisfaction. Few patients present for revisional procedures, and even fewer present for a secondary or repeated reduction mammaplasty. This study defines secondary reduction mammaplasty as performing an additional reduction using a pedicled nipple-areola complex. Few reports of secondary reduction are found in the literature. Operative guidelines for secondary reduction mammaplasty have been published recently. However, the experience of others has differed from these guidelines, and herein is presented another experience with secondary reduction mammaplasty. Ten cases of secondary reduction over a 37-year period were identified and reviewed. The initial reductions were performed using six different techniques. An average of 307 g of tissue per breast (range, 130 to 552 g) was removed at the initial operations. The secondary reductions were performed using four different techniques, and an average of 458 g of tissue per breast (range, 147 to 700 g) was removed at the secondary operations. Three of the 10 patients underwent initial and secondary reduction with the same technique. An average of 4 years (range, 1 to 10 years) separated these surgeries. Seven of the 10 patients underwent initial and secondary reductions with different technique. An average of 15 years (range, 5 to 19 years) separated these procedures. There was an average 5-year follow-up (range, 1 to 20 years) in this series. Four of the 10 patients experienced self-limiting complications after secondary reduction, including delay in wound healing, delay in the return of nipple sensitivity, and mild fat necrosis. Three of the four patients with complications had undergone secondary reduction with a different pedicle technique. No significant or long-lasting skin, pedicle, or nipple-areola complex compromise was found after secondary reduction mammaplasty. In contrast to the recently published guidelines, this study demonstrates that secondary reduction mammaplasty is a safe and viable option when performed with either similar or different technique. This finding allows secondary reduction mammaplasty to be tailored to the individual breast type and to the abilities of the specific surgeon.     

Identification of synergistic interactions among microorganisms in biofilms by digital image analysis.

Digital image analysis showed that reductions in biofilm plating efficiency were due to the loss of protection provided by two benzoate-degrading strains of Pseudomonas fluorescens. This loss in protection was due to the spatial separation of the protective organisms from benzoate-sensitive organisms during the dilution process. Communities were cultivated in flow cells irrigated with trypticase soy broth. When the effluent from these flow cells was plated on 0.15% benzoic acid, satellite colonies formed only in the vicinity of primary colonies. A digital image analysis procedure was developed to measure the size and spatial distribution of these satellites as a function of distance from the primary colony. The size of satellites served as a measure of growth, and the number per unit area served as a measure of survival. At the three dilutions tested, the size and concentration of satellite colonies varied inversely with distance from the primary colonies. When these measurements were plotted, the slopes were used to quantify the effect of bacterial association on the growth and survivability of the satellites. In the absence of the primary colonies, satellites grew in axenic culture only at low benzoate concentrations. Thus benzoate-degrading organisms are capable of creating a protective microenvironment for other members of biofilm communities.